Vesicles were first described in the 1960s as a model of cellular membranes (see Bangham et al., J. Mol. Biol. 13:238-252, 1965). Vesicles have found a number of applications in the delivery of small molecule drugs, vaccine adjuvancy, gene transfer and diagnostic imaging (e.g., see Liposome Technology, 3rd Edition, Edited by Gregory Gregoriadis, Informa HealthCare, 2006 and Liposomes: A Practical Approach (The Practical Approach Series, 264), 2nd Edition, Edited by Vladimir Torchilin and Volkmar Weissig, Oxford University Press, USA, 2003).
A number of methods for preparing vesicles have been described (e.g., see references cited above and Walde and Ichikawa, Biomol. Eng., 18:143-177, 2001). However, there remains a need in the art for methods that can be used to entrap substances within vesicles.
One method that has been described in the art is the so-called 3-step melt method. Vesicle-forming lipids are initially melted at high temperatures (e.g., 120° C.). An emulsion is created in a second step by adding an aqueous buffer (e.g., bicarbonate buffer) to the molten lipids. Finally, the substance to be entrapped is homogenized with the components of the emulsion at a reduced temperature (e.g., 50° C.) prior to lyophilization. Alternatively, vesicles from the emulsion are lyophilized and then reconstituted in the presence of the substance to be entrapped.
While methods such as this one may well be suitable for entrapping substances that can withstand high temperatures and/or small molecules that are able to diffuse rapidly into empty vesicles we have found that they are unsuitable for entrapping the types of antigens (e.g., polypeptides, viruses, etc.) that are commonly involved in vaccines. In particular, we have found that these methods produce low entrapment efficiencies and can dramatically reduce the activity of the underlying antigen (e.g., as measured by immune responses). There is therefore a need in the art for methods of preparing vesicles that are capable of entrapping antigens while minimizing impact on antigen activity.